ABSTRACT The long-range goal of this project is to understand the biological role of corneal crystallins in corneal physiology and pathophysiology. By definition, corneal crystallins are cytosolic enzymes/proteins expressed in high abundance and in a taxon-specific manner in the cornea. The first corneal protein to be identified as a corneal crystallin was aldehyde dehydrogenase 3A1 (ALDH3A1), and several other proteins, including transketolase, ALDH1A1, isocitrate dehydrogenase, and glutathione S-transferase, have subsequently been similarly categorized. We and others have shown that corneal crystallin expression is markedly up-regulated during development and differentiation, specifically as cells exit the cell cycle. Conversely, corneal injury (which triggers cell proliferation) is associated with a loss in expression of corneal crystallins and an increase in light scattering related to corneal haze. We have found that the over-expression of corneal ALDH3A1 causes a profound retardation in cell proliferation, decreased light scattering in vitro and protection against oxidative stress. Our new preliminary data indicate that ALDH3A1 affects cell proliferation and differentiation through both enzymatic and non- enzymatic mechanisms. Our recently-developed congenic Aldh3a1 knockout mice exhibit corneal haze or clouding, analogous to lens cataracts, an observation that confirms our long-standing hypothesis that crystallins are critical to the maintenance of cellular transparency. This is the first genetic animal model of cellular-induced corneal 'haze'. Our working hypothesis remains the same and contends that corneal crystallins (specifically, ALDHs) regulate cell growth, differentiation and cellular transparency through metabolic (enzymatic) and/or structural functions. We now propose a systems biology approach in our novel animal models to identify the molecular mechanism involved in regulating corneal transparency. We will employ state-of-the-art methods in order to quantify changes in tissue structure and function associated with ALDH3A1 expression, including: (a) RNA-sequencing, (b) matrix-assisted laser desorption ionization imaging mass spectrometry (to directly measure metabolites and proteins in situ), (c) integrated pathway analysis, and (d) immunofluorescent tomography (to develop three-dimensional biological mapping of biomolecules within the cornea).